In the rapidly growing field of synthetic biology the cost of synthetic DNA for gene synthesis has become a substantial part of many laboratory budgets. Current DNA synthesis technologies are unable to produce gene length DNA fragments and rely on expensive and error prone assembly methods to construct long strands of DNA. In addition, current DNA synthesis methods are organic chemistry based and produce toxic waste mixtures that are difficult and costly to dispose of. Here we propose to design a novel biologically based DNA synthesis method which will enable the high fidelity, template independent, synthesis of long (>500bp) strands of DNA. This proposal describes a novel method that will lead to reduced costs in every synthetic biology laboratory, enabling applications such as faster development of vaccines, biomolecular computation, reprograming of cells and improved cellular therapeutics. The resulting massively parallel synthesis capability developed in Phase II of this project will be put to use as a custom synthesis service by Molecular Assemblies similar to how custom oligos are ordered, produced and delivered today. To achieve this, this proposal focuses on engineering the enzyme terminal deoxynucleotidyl transferase (TdT), which acts by adding nucleotides to single stranded DNA in a template-independent fashion, to utilize modified deoxynucleotide triphosphates (dNTPs). The dNTP analogs are blocked in such a way that leads to the addition of one and only one nucleotide of choice at a time and, after being added to the growing strand, are able to be de-blocked to regenerate a natural DNA strand. Phase I covers the development of an engineered enzyme and its use to prove the ability to make a short sequence specific nucleic acid. Phase II will lead to optimization of all four dNTP analogs, cycle conditions, automation and the demonstration of the full capabilities of this novel, synthetic approach for polydeoxynucleotides. In 1981 it would have been difficult to envision the specific fundamental roles DNA synthesis would eventually play in modern biology; while the idea of purchasing synthetic genes was a concept of science fiction. One can only imagine how on-demand, high purity, low cost polynucleotides will enable a new era of biological & clinical applications.